Progressive decreases in lysosomal function have been linked to a number of diseases. One cause of decreased function is build-up of indigestible material within lysosomes, which can produce dysfunction. This link is clearest in genetic lysosomal storage disorders but is also apparent with other diseases. The occurrence of lysosomal malfunction increases with age and can be the result of secondary effects creating imbalances in cell homeostatic mechanisms and metabolism. Both innate and acquired lysosomal storage disorders almost always have pathologic consequences and are often fatal. Recently, there has been a growing understanding of the importance of lysosomes in diseases of cholesterol metabolism, such as Niemann-Pick type C, Alzheimer's and most notably atherosclerosis. Lysosomal sterol accumulation in macrophage foam cells is a ubiquitous but not well-understood aspect of atherosclerosis. Macrophages in culture incubated with atherogenic particles also exhibit an accumulation of both free and esterified cholesterol in lysosomes. Our studies show that, initially, free cholesterol accumulates as a result of normal lysosomal hydrolysis of lipoprotein-derived cholesteryl ester but the generated free cholesterol remains trapped in the lysosomes. Subsequent to the free cholesterol accumulation, cholesteryl ester hydrolysis is inhibited and so undegraded lipoprotein cholesteryl ester also accumulates in the lysosomes. Our preliminary data suggests that the inhibition of hydrolysis is mediated, at least in part, through the induction of an inability of lysosomes to maintain an acidic pH. The proposed studies systematically investigate the mechanism by which sterol accumulation inhibits the lysosome's ability to maintain a low pH and tests the hypothesis that free cholesterol, at least partially, mediates the inhibition. In aim 1, cells and isolated lysosomes are used to determine the ability of specific lipids to 1) inhibit pumping of protons into lysosomes, 2) alter the synthesis, stability and trafficking of lysosome proton pumps, 3) increase leakiness of lysosome membranes and 4) whether hydrolysis of other substrates is affected. The lipid content of lysosomes will be altered by incubation with lipoprotein or other lipid-containing particles similar to those found in atherosclerotic lesions. Aim 2 directly modulates sterol, triglyceride, and sphingolipid content of lysosomes ex-vivo to determine if any or all of these affect the lysosomes ability to maintain acidity. Aim 3 explores whether the inability of lysosomes to maintain an active pH is related to cholesterol-mediated alterations in membrane order. Finally, aim 4 investigates whether intra-lysosomal oxidation of lipid explains the trapping of free cholesterol within lysosomes and/or the subsequent inhibition of CE hydrolysis. PUBLIC HEALTH RELEVANCE: Establishing the mechanism and consequences of lysosomal sterol accumulation in foam cells will increase our understanding of atherosclerosis lesion development. The research also has broader implications for other sterol-modulated diseases such as Niemann-Pick and Alzheimer's.